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Abstract

Rapid eye movement (REM) sleep behavior disorder (RBD) is a neurological disease characterized by loss of normal REM motor inhibition and subsequent dream enactment. RBD is clinically relevant because it predicts neurodegenerative disease onset (e.g., Parkinson's disease) and is clinically problematic because it disrupts sleep and results in patient injuries and hospitalization. Even though the cause of RBD is unknown, multiple lines of evidence indicate that abnormal inhibitory transmission underlies the disorder. Here, we show that transgenic mice with deficient glycine and GABA transmission have a behavioral, motor, and sleep phenotype that recapitulates the cardinal features of RBD. Specifically, we show that mice with impaired glycine and GABAA receptor function exhibit REM motor behaviors, non-REM muscle twitches, sleep disruption, and EEG slowing—the defining disease features. Importantly, the RBD phenotype is rescued by drugs (e.g., clonazepam and melatonin) that are routinely used to treat human disease symptoms. Our findings are the first to identify a potential mechanism for RBD—we show that deficits in glycine- and GABAA-mediated inhibition trigger the full spectrum of RBD symptoms. We propose that these mice are a useful resource for investigating in vivo disease mechanisms and developing potential therapeutics for RBD.

Introduction

Rapid eye movement (REM) sleep is paradoxical because muscle tone and movement are largely absent, but overall brain activity is maximal (Jouvet, 1967). The disconnection between the REM-active brain and skeletal motor system may function to ensure motoric quiescence during periods of unconsciousness. The mechanisms that allow the REM-active brain to disconnect itself from the motor system are unknown. Determining how muscle tone is controlled during sleep is clinically relevant because abnormal REM motor control is the defining feature of human REM sleep behavior disorder (RBD).

Patients with RBD are neurologically and motorically normal during waking—it is only during sleep that their primary disease symptoms emerge (Mahowald and Schenck, 2005a). RBD is typified by REM motor activation and violent dream enactment; however, muscle twitches and limb jerks during non-REM (NREM) sleep, sleep disruption, and slowing of the EEG are also defining disease symptoms (Sforza et al., 1997; Olson et al., 2000; Schenck and Mahowald, 2002).

RBD is clinically important for two reasons. First, REM motor activation and repetitive NREM muscle jerks disrupt sleep continuity, and violent dream enactment often results in bodily injuries—lacerations, fractures, and hospital visits are common in RBD (Schenck et al., 1986). Second, a majority of RBD patients develop Parkinson's, multiple system atrophy, or dementia with Lewy bodies within 12 years of initial diagnosis (Schenck et al., 1996; Iranzo et al., 2006). RBD is therefore a harbinger of neurodegenerative disease, particularly those stemming from synucleinopathies.

There is no genetic model of RBD, but such a resource would advance investigation of disease cause and progression. The current study was designed to determine whether genetic impairment of inhibitory neurotransmission could trigger RBD symptoms. To do this, we used infrared videography and electrophysiology to record sleep–wake behavior, motor activity, and muscle tone in transgenic mice with deficient glycine and GABAA receptor function. We show that these mice have a behavioral, motor, and sleep phenotype that mimics human RBD. We propose that abnormal glycine and GABA function could, at least in part, underlie human RBD symptoms. We assert that these transgenic mice could be a valuable resource for pinpointing RBD disease mechanisms.

Materials and Methods

Animals

Transgenic mice were previously generated for determining how reduced glycine and GABA inhibition affects the startle response (Becker et al., 2002). Transgenic mice were engineered to express a mutant glycine receptor α1 subunit 271Q controlled by the Thy-1 promoter. The mutation substitutes a glutamine for an arginine at position 271 in the extracellular domain of the glycine receptor α1 subunit. Mutant receptors are expressed throughout the transgenic CNS. In vitro cell recordings show that transgenic spinal neurons not only experience a 70% reduction in glycine receptor-mediated inhibition, but also exhibit a 91% reduction in GABAA receptor-mediated inhibition. The exact cause of reduced GABAA-mediated inhibition is unknown. Therefore, the transgenic mice used in this study experience a potent reduction in both glycine- and GABAA-mediated inhibition (Becker et al., 2002).

Current experiments used 23 male transgenic mice (23 ± 0.3 g) and 23 male wild-type littermates (29 ± 0.6 g). Mouse lines were bred and handled as previously described (Becker et al., 2002). Animals were both phenotyped and genotyped. Transgenics were visually phenotyped by holding them by the tail, which resulted in clenching of the hindfeet (Becker et al., 2002). In contrast, wild types spread their hindfeet when held by their tails. Transgenic (n = 8) and wild-type (n = 8) mice were also genotyped using PCR (35 cycles, 55°C annealing temperature) with glycine receptor α1-specific primers designed based on the endogenous murine receptor (5′-TGCAAAACCCACAAGAACAA-3′ and 5′-TGGCATTTGTAAGGGTGTGA-3′; common to both wild-type and transgenic animals) and mutant human receptor (5′-TATTCCCAGCCTGCTCATTG-3′; 5′-CGCCCTTGACTGAGATGCCA-3′; only present in transgenic mice). All procedures and experimental protocols were approved by the Animal Care Committee at the University of Toronto and were in accordance with the Canadian Council on Animal Care.

Surgical implantation of EEG and EMG electrodes

Mice were anesthetized with isoflurane (1–2%) and then implanted with EEG and EMG electrodes as described previously (Burgess et al., 2010). EEG recordings were obtained using four stainless steel microscrews (1 mm anterior ±1.5 mm lateral to bregma; 3 mm posterior ±1.5 mm lateral to bregma). EMG electrodes were made from multistranded stainless steel wires (AS131, Cooner Wire), which were sutured onto masseter, hindlimb, and neck muscles. All electrodes were attached to a microstrip connector (CLP-105-02-L-D, Electrosonic), which was affixed onto the animal's head with dental cement (Ketac-cem, 3M). After surgery, mice were given ketoprofen (3 mg/kg) and 5% dextrose in saline. Mice were individually housed in a sound-attenuated and ventilated chamber on a 12:12 light–dark cycle [110 lux; lights on 0700h (7:00 A.M.), lights off 1900h (7:00 P.M.)] for 10–15 d after surgery. Food and water were available ad libitum.

Data acquisition and experimental design

Electrophysiological recordings.

EEG and EMG activity were recorded by attaching a light-weight cable to the plug on the mouse's head, which was connected to a Super-Z head-stage amplifier and BMA-400 AC/DC Bioamplifier (CWE). The EEG was amplified 1000 times and bandpass filtered between 1 and 100 Hz. EMG signals were amplified 1000 times and bandpass filtered between 30 and 500 Hz. All electrophysiological signals were digitized at 1000 Hz (Spike 2 Software, 1401 Interface, CED) and monitored and stored on a computer. Infrared video recordings were captured (Sony DCR-HC28) and synchronized with the electrophysiological recordings using Spike 2 software.

Experimental protocols.

At the start of each experiment, animals were transferred to a round Plexiglas cage (diameter: 20 cm) inside a sound-attenuated, ventilated, and illuminated (110 lux) chamber. Each mouse was given 24 h to habituate to this environment. Animals were then tethered, using a lightweight tether attached to the Raturn system (BAS). Mice were given another 24 h to habituate to the recording tether. After habituation, we recorded 24 h of undisturbed video, EEG, and EMG activity.

Drug preparation and treatment

Clonazepam.

Clonazepam is a benzodiazepine that is used to treat human RBD. It rapidly alleviates RBD motor symptoms in 90% of patients (Lapierre and Montplaisir, 1992; Schenck et al., 1993; Olson et al., 2000). To investigate the effects of clonazepam on the motor phenotype of transgenic mice, a subset of wild-type and transgenic mice received intraperitoneal injections of 0.3 mg/kg clonazepam (Roche) dissolved in 0.9% saline. All injections were administered at 1:00 P.M. The volume of drug given to each animal was determined before each injection based on the animal's weight, and this volume was topped up with saline such that each animal received 0.3 mg/kg clonazepam in a 0.2 ml bolus. EEG and EMG activity were quantified for the 3 h following the injection. Effects of clonazepam on sleep and motor activity were compared to each animal's own pretreatment levels.

Melatonin.

Long-term melatonin treatment is used to alleviate RBD symptoms (Takeuchi et al., 2001; Boeve et al., 2003; Kunz and Mahlberg, 2010). To chronically treat wild-type and transgenic mice, a melatonin (Sigma-Aldrich) solution was prepared in ethanol and dissolved in the drinking water (12.5 μg/ml tap water, in 0.07% ethanol) for 2–4 weeks. The water bottle containing melatonin was protected from light throughout the experiment. A fresh melatonin solution was prepared twice per week. Both wild-type and transgenic mice drank an average of 4 ± 0.3 ml per 24 h; this volume is consistent with a previous report (Johnson et al., 2003). Although the volume of water intake varied among animals, melatonin was administered at ∼2 mg/kg/d. A group of wild-type and transgenic mice were treated with melatonin for 2–4 weeks, and then EEG and EMG activity was quantified over a 24 h period. This activity was compared to their untreated counterparts.

Sleep–wake architecture.

The proportion of time spent in each sleep–wake state was calculated across a 24 h period and compared between wild-type and transgenic mice. The number of state transitions (i.e., arousals from NREM and REM sleep, NREM to REM transitions) was also quantified.

EEG spectral analysis.

EEG spectral analysis was calculated in 1 Hz bins using fast Fourier transformation of each 5 s epoch, yielding a power spectra profile from 0 to 16 Hz. A mean EEG spectrum profile was obtained for each epoch and then, to minimize nonspecific differences in absolute power between individuals, EEG power in each frequency bin was expressed as a percentage of the total EEG power in the epoch. The spectral profiles of each behavioral state were then compared between wild-type and transgenic mice.

Statistical analyses

All statistical analyses used SigmaStat (SPSS) and applied a critical α value of 0.05. In all comparisons, the Kolmogorov–Smirnov test was used to test for normality. Differences in EMG activity between wild-type and transgenic mice were determined using t tests (or Mann–Whitney rank sum test for nonparametric data). The proportion of time spent in each sleep–wake state and total EEG power in each state were compared between wild-type and transgenic mice using ANOVA with repeated measures (RM ANOVA), and post hoc comparisons were performed using the Student–Newman–Keuls (SNK) test. All data are expressed as mean ± SEM.

We found that 100% of transgenic mice (n = 16) exhibited motor behaviors that mimic RBD symptoms. During REM sleep, mutants displayed gross body and limb movements—running, jerking, and chewing were common behaviors. Even though uncoordinated muscle twitches and jerks occurred, most motor events were characterized by coordinated limb and head movements. Such motor behaviors influenced sleep posture. Unlike wild-type mice, which slept in a typical curled position, transgenics slept on their side during REM sleep. Overt motor behaviors occurred throughout REM sleep episodes; however, there were also defined periods of motor quiescence. Motor behaviors and generalized body movements occurred during all REM episodes in all transgenic mice.

Because REM behaviors were wake-like in nature, we wanted to verify that such behaviors occurred during bona fide REM sleep episodes and not during waking. First, we showed that REM sleep is electrophysiologically different from wakefulness in transgenics. Quantitative spectral analysis demonstrates unequivocal differences in EEG frequencies between identified periods of wakefulness and REM sleep in transgenic mice (waking vs REM: RM ANOVA, F(1,14) = 14.71, p < 0.001) (Fig. 3A). We also found that REM sleep episodes were significantly shorter than periods of wakefulness (Mann–Whitney, U = 134.0, n1 = n2 = 16, p < 0.001) (Fig. 3B). Second, we showed that REM sleep characteristics (except motor activation) are similar between transgenic and wild-type mice. We found that transgenic and wild-type mice spend the same amount of time in REM sleep (SNK, q = 0.63, p = 0.658) (Fig. 3C), and we showed that REM sleep EEG frequencies are similar in the two types of mice (Fig. 3D).

Verification that RBD behaviors occur during REM sleep. Because RBD behaviors are reminiscent of waking activity, we used post hoc analyses to confirm that such behaviors occur during REM sleep. A, In transgenic mice (Tg; n = 16), we show that there is a marked difference in the distribution of spectral power between periods of identified wakefulness and REM sleep. B, We also show that the length of identified REM sleep episodes is significantly shorter than periods of waking. C, Compared to wild-type mice (Wt; n = 19), transgenic mice spend the same amount of time in REM sleep. D, Also similar to wild-type mice, EEG spectral power is concentrated in the theta range during periods of identified REM sleep in transgenic mice. *p < 0.001. All values are mean ± SEM.

Transgenic mice exhibit RBD symptoms during NREM sleep

Repetitive muscle twitches/jerks during NREM sleep, especially in the limbs, are also a cardinal feature of RBD (Schenck and Mahowald, 2002). Therefore, we aimed to determine whether transgenic mice experience abnormal motor activity during NREM sleep. We found that 100% of transgenic mice (n = 16) experienced brief, repetitive EMG twitches and limb jerks during NREM sleep (Fig. 5). Twitches generally occurred simultaneously in each recorded muscles and were of sufficient magnitude to cause overt body movement and postural changes. This motor behavior is in sharp contrast to NREM activity in wild-type littermates, which is characterized by complete motor quiescence and inactivity.

Sleep is disrupted in transgenic mice. A, Sleep disruptions experienced by transgenic mice (Tg; black bars, n = 16) increase the amount of wakefulness and decrease NREM sleep in these mice compared to their wild-type littermates (Wt; white bars, n = 19). REM sleep amounts are unchanged. The increase in wakefulness is due to an increase in the amount of time spent in quiet wake (inset). B, The decrease in NREM sleep is due to a decrease in the length, not number, of NREM episodes. *p < 0.01. All values are mean ± SEM.

EEG slowing in transgenic mice. EEG spectral profiles for wild-type (n = 15; dotted line) and transgenic (n = 11; solid line) mice for wake (A) and NREM (B) sleep. Transgenics have more power in the lower frequency ranges and less power in the higher frequency ranges, resulting in an overall EEG slowing in these states. *p < 0.01. All values are mean ± SEM.

Clonazepam and melatonin rescue the RBD phenotype

Clonazepam is the most common and effective treatment for RBD (Schenck and Mahowald, 2002). Clonazepam functions to strengthen GABAergic inhibition by acting on benzodiazepine receptors. Even though GABAA-mediated inhibition is reduced in transgenics, they nonetheless have functional GABAA receptors (Becker et al., 2002). Therefore, we wanted to determine whether clonazepam could strengthen inhibition and thereby rescue the RBD phenotype in mutant mice.

Discussion

RBD is a public health concern because it forecasts neurodegenerative disease, disrupts sleep, and results in patient injuries and hospitalization (Mahowald and Schenck, 2005a). Transgenic mice with deficient glycine and GABAA receptor function are the first model of RBD that successfully recapitulates the behavioral, motoric, and sleep features that define the disorder—i.e., REM behaviors, NREM myoclonic jerks, sleep fragmentation, and EEG slowing (Table 1). Importantly, the RBD phenotype in transgenic mice can be rescued by drugs that treat human disease symptoms. Our findings are the first to indicate that deficits in glycine- and GABAA-mediated inhibition trigger the full spectrum of RBD symptoms. We propose that these mice are a powerful resource for investigating in vivo disease mechanisms and developing potential therapeutics for RBD.

Even though RBD has multiple triggers, a common mechanism could nonetheless underlie the disorder. Our current results show that reduced glycine- and GABAA-mediated inhibition induces RBD motor symptoms in transgenic mice. Accordingly, we assert that impaired inhibitory neurotransmission plays a central role in triggering RBD motor behaviors during sleep. Multiple lines of clinical and experimental evidence support this assertion.

Sleep is disrupted in human RBD. Patients not only arouse more from sleep, they can also experience more stage 1 NREM sleep (Schenck et al., 1987; Sforza et al., 1988). REM motor behaviors and NREM limb jerks may elicit sleep disruption; however, most arousals from sleep are not associated with preceding motor activity (Schenck et al., 1993). Current results show that most arousals from sleep (i.e., 90%) are not caused by NREM motor activity in transgenic mice, suggesting that other factors elicit sleep disruption in RBD. Because glycine and GABA regulate normal sleep and muscle tone, we assert that sleep disruption in RBD is caused not only by motor events, but also by abnormal inhibitory transmission. This hypothesis is supported by the fact that drugs that enhance inhibition also improve sleep in both transgenic mice and RBD patients (Kunz and Bes, 1999).

Increasing inhibitory tone improves RBD symptoms

Drugs that strengthen inhibitory activity effectively alleviate RBD symptoms. Clonazepam and melatonin are the most common treatments for RBD (Schenck and Mahowald, 2002)—both function to enhance GABAergic transmission (Skerritt and Johnston, 1983; Coloma and Niles, 1988; Rosenstein et al., 1989; Wu et al., 1999). We found that clonazepam and melatonin also alleviated RBD symptoms in transgenic mice. Both drugs reduced motor activation and behavior during REM sleep, but only clonazepam suppressed NREM muscle twitches/jerks. We also showed that melatonin, but not clonazepam, effectively improved sleep in mutant mice. These drugs may improve motor function and sleep by restoring GABAergic tone to sleep and motor circuits that are deficient in transgenic mice. Because melatonin and clonazepam have differential effects on sleep and motor function, each may act at unique sites within the brainstem circuitry.

RBD motor symptoms typically worsen with time (Iranzo et al., 2009b), suggesting that progressive neuronal degeneration contributes to RBD symptoms. While there is no clear evidence that dopaminergics influence RBD symptoms (Iranzo et al., 2009a), imaging studies nonetheless indicate that dysfunction of the nigrostriatal dopamine system is associated with RBD (Albin et al., 2000; Eisensehr et al., 2000). We suggest that initial RBD onset is triggered by loss of GABAergic/glycinergic function and that progressive dopamine cell degeneration not only causes Parkinson's symptoms but also worsens RBD.

A mouse model of RBD

The underlying cause of RBD is unknown. Current results show that impaired inhibitory transmission triggers the hallmark features of RBD in transgenic mice. Although our results show that abnormal glycine/GABAA receptor activity elicits an RBD phenotype, other proteins mediating inhibition (e.g., potassium chloride transporter 2, KCC2) could also be involved in triggering RBD symptoms. Determining the specific inhibitory mechanism(s) underlying RBD is therefore of immediate clinical importance.

Footnotes

This study was funded by the Canadian Institutes of Health Research, the National Science and Engineering Research Council of Canada, the Canadian Foundation for Innovation, and the Parker B. Francis Foundation. We thank Dr. Hans Weiher for donating the founder line of transgenic mice. We also thank University of Toronto support staff for providing animal care.

Correspondence should be addressed to Dr. John Peever, Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, Ontario M5S 3G5, Canada.John.Peever{at}utoronto.ca

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